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. 2019 Jul 30;9:653. doi: 10.3389/fonc.2019.00653

Figure 2.

Figure 2

miR-326 targets multiple genes in ErbB/PI3K pathway. (A) RT-qPCR results of the expression of potential targets (EGFR, ErbB2, ErbB3, AKT1, AKT2, and AKT3) upon miR-326 and mock vectors transfection in SKBR3 cells. (B) Relative luciferase activity of reporter plasmids carrying the wild-type UTR or a fragment lacking any binding site for miR-326 (as a control UTR) in HEK293 cells co-transfected with miR-326 or mock vector. (C) Expression level of phospho-Akt and total AKT protein were detected by Western blotting in miR-326 and mock transfected SKBR3 cells. (D) ErbB2, ErbB3, and AKT1 expression levels in SKBR3 cells transfected with ErbB2 psicheck or co-transfected with ErbB2 psicheck and mock or miR-326 were significantly higher than the cells transfected with control vectors (mock, psicheck empty vector, and combination of mock and psicheck vectors) that indicates ErbB2 rescued miR-326 target genes (ErbB2, ErbB3, and AKT1 genes). In contrast, ErbB2, ErbB3, and AKT1 expression levels in SKBR3 cells transfected with miR-326 or co-transfected with psicheck empty vector and miR-326 were significantly lower than the cells transfected with control vectors (mock and combination of mock and psicheck vectors). Assays were performed in triplicate. Means ± SEM was shown. Statistical analysis was conducted using student t-test.